Analyses of the anthrax toxin proteins by our group and others continues to generate opportunities to engineer the proteins as tools for cell biology and as the basis of vaccines and therapeutics. Our prior work provided robust methods for production and purification of the anthrax toxin proteins: protective antigen, lethal factor, and edema factor. Many different substitution mutants and fusion proteins have been constructed to facilitate analysis and exploitation of these proteins. We previously arranged through the NIH technology transfer office for many of these proteins to be distributed by Kerafast. Their website lists more than 10 of our protein variants that are available to researchers wishing to evaluate their utility. Kerafast also continues to distribute the unique S9.6 monoclonal antibody that recognizes DNA/RNA hybrids. This antibody was also licensed to EMD/Millipore. This antibody remains extremely popular, being sold to hundreds of researchers for analysis of R-loops, structures in which nascent mRNA is bound to one DNA strand, displacing the opposite DNA strand, producing what is termed an R-loop. We continued efforts to produce useful amounts of a single chain (scFv) version of the antibody. Our studies on protein expression and characterization have been extended during 2017 to additional proteins produced by several bacterial pathogens. A number of expression systems have been developed for production of the mycobacterial proteins ESAT6 and Ag85b. These are being engineered with modifications that will allow them to be used as vaccine candidates and immune modulators. A key use of our knowledge of anthrax toxin structure is in design of tumor-targeting agents. In prior work we generated toxin variants that depend on activation by two cancer cell surface proteases, matrix metalloproteases (MMP) and urokinase plasminogen activator (uPA). These agents contain two separate proteins, each activated by one of the two proteases. The design requires that the two mutated and inactive proteins must combine to achieve activity. We have designated these as intercomplementing protective antigen drugs, or IC-PA. By requiring activation by two proteases, activity toward normal tissues is greatly decreased, and therapeutic indices are increased. In 2017 we created a new type of IC-PA based on mutations to amino acids that interact in the lumen of the protective antigen pore during conversion of the oligomeric pre-pore to an active pore. During this PA pre-pore-to-pore conversion, the intermolecular salt bridge interactions between Lys397 and Asp426 on adjacent PA protomers play a critical role in positioning neighboring luminal Phe427 residues to form the Phe-clamp, an essential element of the functional PA pore. This essential intermolecular interaction afforded the opportunity to create pairs of PA variants that depend on intermolecular complementation to form a functional pore. We found that PA-U2-K397Q, a urokinase-activated PA variant with Lys397 residue replaced by glutamine, and PA-L1-D426K, a MMP-activated PA variant with Asp426 changed to lysine, form functional pores only when administered together. Further, the mixture of PA-U2-K397Q and PA-L1-D426K displayed potent anti-tumor activity in the presence of LF. Thus, PA-U2-K397Q and PA-L1-D426K form a novel intermolecular complementation system (termed IC4-PA) with toxin activation relying on the presence of two distinct tumor-associated proteases, i.e., urokinase and MMPs. While IC4-PA uses a novel mechanism to achieve the intercomplementation effect, it is not clearly superior to the earlier versions, IC2-PA and IC3-PA. Thus, our attempts to move these agents into clinical use will focus on the best-studied agent, IC3-PA. We are continuing to characterize its mechanism of action, optimal dose schedule, and efficacy in additional mouse tumor models. In addition, we are actively seeking partners to expand existing trials in veterinary canine and feline cancer patients.